Hemopoiesis involves the active process of proliferation and differentiation of pluripotent progenitor cells into all types of mature blood cells and some specialized tissue cells. Production of functional blood cells is regulated by specific proteins, the hemopoietic growth factors (HGFs). Some of the HGFs control maturation of a specific maturation lineage, whereas others stimulate proliferation and differentiation of progenitors along multiple pathways. Much of our knowledge of the hemopoietic differentiation process has been obtained from mouse studies in vitro and in vivo, using purified growth factors. The murine growth factor interleukin-3 (mIL-3), also termed multi-CSF, mast cell growth factor, stem cell activating factor or several other designations, stimulates the proliferation of developmentally early, multipotent cells (CFU-S) as detected by the spleen colony assay, resulting in the production of progenitor cells along the erythroid, megakaryocyte, granulocyte/macrophage, osteoblast and several other lineages. Furthermore, mIL-3 has been implicated in replication of pluripotent stem cells, probably in synergism with other HGFs.
In recent years, several groups have succeeded in cloning mIL-3 cDNA. No results have been reported so far of identifying homologous sequences in human DNA using mIL-3 DNA as a probe. Presumably, the human gene has diverged extensively from the mIL-3 gene or has lost its function during primate evolution. However, human leukocytes were found to produce an HGF(s) which can replace M-GSF in supporting the proliferation of murine CFU-S. Thus, the existence of a human HGF was postulated, which shares biological properties with mIL-3 and therefore could be the human homolog.
Recently, DNA sequences encoding hIL-3 have been identified by several investigators. For instance, using as probe a cDNA coding for gibbon IL-3, the human IL-3 gene was isolated (Yang et al., 1986). The sequence of the exons of the human gene was disclosed in the cited paper as well as in patent application WO 88/00598 (published 28 Jan. 1988). However, as known to those skilled in the art, the intron-containing genomic sequence cannot be used for synthesis of hIL-3 in microorganisms. Rather, the coding sequence used should be a continuous coding sequence as in a cDNA. A cDNA sequence encoding human IL-3 is also disclosed in WO 88/00598. Following another route, Dorssers et al. (1987) also isolated a cDNA coding for human IL-3.
Patent Application No. WO 88/05469 discloses the isolation of a cDNA encoding hIL-3 using a synthetic DNA derived from the genomic sequence described by Yang et al. (1986) as a probe. The disclosed cDNA sequence, however, lacks two amino acids, nos. 44 and 45 or 45 and 46. The amino acid bordering either deletion is a GAC encoded Asp. Nonetheless, the culture supernatant of a yeast transformant carrying this cDNA sequence in an expression cassette, encoding mature hIL-3 fused to an N-terminal "flag" of 8 amino acids, shows IL-3 activity in a human bone marrow proliferation assay. This finding indicates that the absence of the aforementioned two amino acids and the N-terminal extension of 8 amino acids has no deleterious effect on the biological activity of the protein.
Finally, EP 282.185 also discloses the isolation of a hIL-3 cDNA sequence using as probe a synthetic DNA derived from the genomic sequence described by Yang et al. (1986) and describes the construction of a completely synthetic hIL-3 coding sequence as well as the construction of two muteins, Ile.sup.2 and Leu.sup.131. There is no mention of biological activity. Furthermore, it was apparently assumed that hIL-3 contains 132 amino acids, starting at the N-terminus with Pro.sup.1 -Met.sup.2 -, whereas it is generally accepted that hIL-3 is 133 amino acids long and has as the N-terminus Ala.sup.1 -Pro.sup.2 -Met.sup.3.
It is noteworthy that Yang et al. (1986) find a Ser residue at position 8 of the mature hIL-3, whereas all other references indicate the presence of a Pro at this position.
BPV-1 or the 69% subgenomic fragment (BamHI-HindIII) has been used for the expression cloning of a variety of genes in different cloning systems. EP-A-198386 describes the expression of gamma-interferon in C127 mouse cells. In EP-A-105141 the use of the BPV vector is described for the expression of hepatitis B surface antigen (HBsAg) in vertebrate cell lines e.g. NIH 3T3, LTK.sup.- mouse fibroblasts and African green monkey kidney cells. The general idea of using BPV-1 is disclosed in U.S. Pat. No. 4,419,446.
BPV-1 is one of at least six bovine papillomaviruses and is associated with cutaneous fibropapillomas in cattle. These viruses can readily transform a variety of rodent cells in culture. The molecularly cloned bovine papillomavirus DNA as well as a cloned 69% subgenomic fragment are efficient in inducing transformed foci. Transformed cells contain multiple copies (10 to 120 per cell) of the viral DNA as unintegrated molecules (Law et al., 1981). The genetics of bovine papillomavirus type I have been extensively studied (for a review see Lambert et al., 1981). The BPV-1 genome is a circular, 7946 base-pair, double-stranded DNA molecule. The transcription is complicated because of the presence of multiple promoters, splice sites, and differential production of RNA species. The activities of some of the promoters are under tight control of transcriptional enhancers.
The so-called E2 (=early) ORF is very important in this respect. The full-length E2 ORF encodes a transactivating protein (E2-ta) which can stimulate transcription of the early genes.
This protein consists of two conserved domains, the amino terminal domain (which has transactivating activity) and the carboxy-terminal domain (which has both DNA-binding and dimer formation activities). The E2 ORF encodes a second regulatory protein, the E2 transcriptional repressor (E2-tr), which is an amino-terminally truncated form of the E2-ta protein. E2-tr is encoded by another mRNA, whereby the translation initiation codon is an E2 ORF internal ATG-codon.
The present invention discloses cell lines not previously employed in hIL-3 production as well as mutations of the E2 ORF.
The clinical utility of hIL-3 is not only dependent on its inherent characteristics but also on its availability and the lack of contaminants. The prior art relating to the purification of murine, gibbon and human IL-3 is briefly reviewed here.
The mature murine T-cell enzyme marker 20a-hydroxy-steroid dehydrogenase (20aSDH) was found to be inducible in vitro. The factor responsible for this was partially purified from splenic lymphocytes by Ihle et al. (1981). It was distinct from other known lymphokines in both its biochemical and functional properties. Ihle et al. (1981) proposed the term interleukin-3 ("IL-3") for this factor. The purification by Sephadex G-100 and DEAE cellulose chromatography resulted in a 9000-fold purification, yet the final preparation still contained multiple proteins.
An improved purification procedure was presented by Ihle et al. (1982), wherein WEHI-3 cells which constitutively produce IL-3, were used. Here, through the extension of the earlier procedure with hydroxylappatite and reverse-phase high performance liquid chromatography, the final product could be obtained 1,800,000-fold purified (their Table I). This product was claimed to be homogeneous.
Miyajimi et al. (1987) used the silkworm Bombyx mori and an insect baculovirus vector for high-level expression and secretion of murine IL-3. Purification of IL-3 from tissue culture medium was carried out by sequential passage through DEAE-Sephadex, ACA 54 and C8 reverse-phase column chromatography. To obtain separation of three species of IL-3 (18, 20 and 22 kDa) a second C8 reverse-phase column was necessary. The different species are due to differential glycosylation, since N-glycanase treatment yielded one final band of 15 kDa.
Ziltener et al. (1988) described the isolation of multiple glycosylated forms of IL-3 by affinity purification. The observed microheterogeneity was dependent on the source (activated T-cells, WEHI-3B cells or COS 7 cells).
All of the above procedures describe the purification of murine IL-3. In spite of the observed similarity of murine and human IL-3 with respect to their proliferative action on haematopoietic progenitor cells, the structural homology between both proteins is rather low (28% at amino acid level). This heterogeneity is illustrated by the total absence of reactivity of the human protein on murine cells (and vice versa). Based on their specific amino acid composition, the proteins likely require different methods of purification.
The purification of both gibbon and human IL-3, which show a structural homology of 93% (at the amino acid level), is disclosed in several patent publications. WO 88/00598 describes the isolation of a partially synthetic hIL-3 from the inclusion bodies of E. coli cells. The cells are first disrupted by two passages through a french press, and the inclusion bodies are isolated by centrifugation in a sucrose step gradient. This reference describes also three procedures for purifying a human or gibbon IL-3-like polypeptide from COS cell conditioned medium. In all cases a one-column process is used: either ion exchange or a lentil lectin column or reversed-phase HPLC. The maximum purity obtained for gibbon IL-3 as determined with automated Edman degradation was 98%.
WO 88/05469 describes the purification of human IL-3 from yeast strains by single or sequential reversed-phase HPLC steps. Since additional HPLC steps can be employed if indicated, it is clearly not assumed that the product is homogeneous. No test of the purity of the hIL-3 was described, nor were data mentioned on the purity of the product.
Thus, no specific methods have been disclosed so far for the purification of hIL-3. Therefore there is still a need for substantially pure hIL-3 which can be used therapeutically and for a method of preparing such substantially pure product in a high yield, and which can easily be scaled up.